A. Field of the Invention
The present invention relates to microscopy and, in particular, to total internal reflection microscopy (TIRM) and to prism-type variable-angle TIRM (sometimes referred to as VA-TIRM). The invention can include a pseudo total internal reflection microscopy mode. One type of TIRM 15 TIRFM (Total Internal Reflection Fluorescence Microscopy (conventional or pseudo)), which creates an evanescent field (EF) and gathers fluorescence generated by the EF. Another type of TIRM gathers Raman scattering. Another type gathers scattered light from plasmonic materials including nano-particles. Another investigates plasmon surface resonance.
The invention relates to use of scanning single-prism-type TIR microscopy for collection of any of a variety of scattered radiation or another signal, including but not limited to study of fluorescence, surface plasmon scattering spectra, Raman spectra, plasmonic materials or particles, or analogous effects or phenomena.
B. Related Art
TIRFM is a well-known type of TIR microscopy. TIRFM takes advantage of an optical effect that can be adapted to observe fluorescent events occurring at the interface between two optical media of different refractive indices. Excitation light incident upon such a boundary, travelling at an angle greater than the critical angle, undergoes total internal reflection. The electromagnetic field of the total internal reflected light extends into the sample beyond the interface. Extending only several tens of nanometers into the second medium of lower refractive index, typically along the optical axis of the microscope in essentially the z direction, this evanescent field decreases exponentially in intensity along the z-axis of penetration. Only the section of the specimen located within the evanescent field undergoes fluorescence excitation. Although TIRFM is limited to the area within several hundreds of nanometers of the substrate/sample interface where total internal reflection is occurring, it is an increasingly popular technique for visualizing, with high signal-to-noise ratio, nano-scale structures. One particularly important application is visualizing and imaging processes that occur in and around the membrane of living cells (partially due to availability of novel membrane-specific fluorophores). TIRFM is also a common technique for imaging single molecules dynamics.
Two typical TIRFM techniques are known as objective-type and prism-type. The objective-type approach requires that the laser be introduced through the microscope objective, and the angle over which TIR can be practically achieved requires use of an oil-immersion objective (with big numerical aperture or N.A.). Also, the objective configuration can result in limitations of available angle range for incident illumination light. General principles of TIRFM in the context of objective-type TIRFM can be found at U.S. RE38,307, incorporated by reference herein. The present invention relates to prism-type TIRFM.
Prism-type TIRFM illuminates a sample on a prism with an evanescent field layer at the prism/sample interface. Typically a laser light is directed through a prism to the sample, wherein the angle of incidence of the laser results in total internal reflection at that prism/sample interface. An evanescent field layer is generated from the prism/sample interface. (Sometimes, the sample can be put on a glass slide which is put on the top of prism. Then the evanescent field will happen at the slide/sample interface.) The thickness of the field is dependent on the angle of incidence and the frequency of laser light. Adjusting those parameters, structures of a much smaller scale in the sample, within the depth of the evanescent field, can be resolved than with regular light microscopes. The evanescent field technique suppresses background to get better contrast and thus better resolution of smaller features. The higher resolution is substantial compared to other normal light microscopes, e.g., epi-fluorescence, or bright field microscope. General principles regarding prism-type TIRFM can be found at U.S. published patent application US2005/0057798 and U.S. Pat. No. 6,255,642, each incorporated by reference herein.
Electron microscopes are an alternative for resolution of submicron or nanoscale features. However, the systems are quite expensive. Additionally, they require drying and exposure of the sample to vacuum. This prevents such things as observing live cells. Therefore, electron microscopes are not indicated for the study of live cells, which is in increasingly greater demand. Observation of live cells may lead to a better understanding of cells and their functions, and thus lead to substantial advancements, including but not limited to, biology, medicine, and other sciences.
There is a need for better resolution than existing microscopy techniques with respect to many applications which can be flexible, adaptable, effective, and more economical than, for example, electron microscopes. There have been attempts to obtain sub-diffraction-limited spatial information with a different approach than the present invention. See, for example, U.S. published application 2009/0237501, incorporated by reference herein. It describes some of the hurdles as well as some of the benefits and applications of the ability to do so.
The principles of TIRFM are well-known. A variety of manufacturers (e.g. Nikon, Zeiss, Olympus) provide commercially-available objective-type TIRFM systems. Such systems can include not only a light microscope, but also a programmable controller and/or computer that can communicate with such things as electronically-controller mirror turrets, stages, illumination sources, displays, and cameras.
In TIRFM, a sample (e.g. live cells) suspended in a liquid or semi-liquid phase substance) is placed on a glass microscope slide. A source of coherent light is directed at an angle of incidence such that it has total internal reflection (TIR) (the light does not refract and pass through the slide). A standing wave at the slide/sample interface creates a very thin evanescent layer above the interface. By varying angle of incidence (variable angle or VA) of the illuminating light, the thickness of the layer is changed. One can see or resolve through the microscope things at varying vertical (axial to the microscope objective) distances from the interface. Thus, one can not only resolve very small individual particles or features within that layer not possible with a normal light microscope, but can vary the thickness of the layers so features can be resolved at different vertical (axial) distances from the interface surface. Thus, varying angle of incidence of the illuminating light can essentially section the sample on the slide by changing that angle of incidence systematically.
But there are issues with present VA-TIRFM technology. For example, performance of present systems largely depends on the accuracy, precision, and reproducibility of tedious, time-consuming re-calibration procedures before each sample is imaged. This limits the through-put and efficiency of such systems. It also exposes such systems to unintentional human error. The theoretical spatial resolution of evanescent field (EF) excitation has not been harvested because it is difficult or impossible to manually calibrate the system at all relevant incident angles within small margins of error. Also, use of such TIRFM systems for 3D image reconstruction has been hampered by difficulties performing full Laplace transforms or fitting with nonlinear least squares methods. Many TIRFM systems do not have any systematic way to step the excitation plane through the sample. Another issue that arises with TIRFM are limitations regarding illumination depth.
One or more similar issues to those discussed above exist regarding TIR microscopy relevant to Raman scattering, plasmonic scattering, surface plasmon resonance, and analogous effects or phenomena.
Therefore, there is room for valuable improvement in these technical areas.